A 12 000-rad whole-genome radiation hybrid panel in sheep: application to the study of the ovine chromosome 18 region containing a QTL for scrapie susceptibility

Whole-genome radiation hybrid (RH) panels have been constructed for several species, including cattle. RH panels have proven to be an extremely powerful tool to construct high-density maps, which is an essential step in the identification of genes controlling important traits, and they can be used to establish high-resolution comparative maps. Although bovine RH panels can be used with ovine markers to construct sheep RH maps based on bovine genome organization, only some (c. 50%) of the markers available in sheep can be successfully mapped in the bovine genome. So, with the development of genomics and genome sequencing projects, there is a need for a high-resolution RH panel in sheep to map ovine markers. Consequently, we have constructed a 12 000-rad ovine whole-genome RH panel. Two hundred and eight hybrid clones were produced, of which 90 were selected based on their retention frequency. The final panel had an average marker retention frequency of 31.8%. The resolution of this 12 000-rad panel (SheepRH) was estimated by constructing an RH framework map for a 23-Mb region of sheep chromosome 18 (OAR18) that contains a QTL for scrapie susceptibility.     

A high-resolution 15,000(rad) radiation hybrid panel for the domestic cat

The current genetic and recombination maps of the cat have fewer than 3,000 markers and a resolution limit greater than 1 Mb. To complement the first-generation domestic cat maps, support higher resolution mapping studies, and aid genome assembly in specific areas as well as in the whole genome, a 15,000(Rad) radiation hybrid (RH) panel for the domestic cat was generated. Fibroblasts from the female Abyssinian cat that was used to generate the cat genomic sequence were fused to a Chinese hamster cell line (A23), producing 150 hybrid lines. The clones were initially characterized using 39 short tandem repeats (STRs) and 1,536 SNP markers. The utility of whole-genome amplification in preserving and extending RH panel DNA was also tested using 10 STR markers; no significant difference in retention was observed. The resolution of the 15,000(Rad) RH panel was established by constructing framework maps across 10 different 1-Mb regions on different feline chromosomes. In these regions, 2-point analysis was used to estimate RH distances, which compared favorably with the estimation of physical distances. The study demonstrates that the 15,000(Rad) RH panel constitutes a powerful tool for constructing high-resolution maps, having an average resolution of 40.1 kb per marker across the ten 1-Mb regions. In addition, the RH panel will complement existing genomic resources for the domestic cat, aid in the accurate re-assemblies of the forthcoming cat genomic sequence, and support cross-species genomic comparisons.     

ChickRH6: a chicken whole-genome radiation hybrid panel

As a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6 000 rads with HPRT-deficient hamster Wg3hCl₂ cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs.     

Radiation hybrid maps of Medaka chromosomes LG 12, 17, and 22.

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was approximately 33 kb/cR. We estimate the potential resolution of the RH panel to be approximately 186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka.     

Identification and High-Density Mapping of Gene-Rich Regions in Chromosome Group 5 of Wheat

The distribution of genes and recombination in the wheat genome was studied by comparing physical maps with the genetic linkage maps. The physical maps were generated by mapping 80 DNA and two phenotypic markers on an array of 65 deletion lines for homoeologous group 5 chromosomes. The genetic maps were constructed for chromosome 5B in wheat and 5D in Triticum tauschii. No marker mapped in the proximal 20% chromosome region surrounding the centromere. More than 60% of the long arm markers were present in three major clusters that physically encompassed <18% of the arm. Because 48% of the markers were cDNA clones and the distributions of the cDNA and genomic clones were similar, the marker distribution may represent the distribution of genes. The gene clusters were identified and allocated to very small chromosome regions because of a higher number of deletions in their surrounding regions. The recombination was suppressed in the centromeric regions and mainly occurred in the gene-rich regions. The bp/cM estimates varied from 118 kb for gene-rich regions to 22 Mb for gene-poor regions. The wheat genes present in these clusters are, therefore, amenable to molecular manipulations parallel to the plants with smaller genomes like rice.     

A high-resolution comparative RH map of the proximal part of bovine chromosome 1

Current comparative maps between human chromosome 21 and the proximal part of cattle chromosome 1 are insufficient to define chromosomal rearrangements because of the low density of mapped genes in the bovine genome. The recently completed sequence of human chromosome 21 facilitates the detailed comparative analysis of corresponding segments on BTA1. In this study eight bovine bacterial artificial chromosome (BAC) clones containing bovine orthologues of human chromosome 21 genes, i.e. GRIK1, CLDN8, TIAM1, HUNK, SYNJ1, OLIG2, IL10RB, and KCNE2 were physically assigned by fluorescence in situ hybridization (FISH) to BTA1q12.1-q12.2. Sequence tagged site (STS) markers derived from these clones were mapped on the 3000 rad Roslin/Cambridge bovine radiation hybrid (RH) panel. In addition to these eight novel markers, 17 known markers from previously published BTA1 linkage or RH maps were also mapped on the Roslin/Cambridge bovine RH panel resulting in an integrated map with 25 markers of 355.4 cR(3000) length. The human-cattle genome comparison revealed the existence of three chromosomal breakpoints and two probable inversions in this region.     

Characterization of a YAC and cosmid contig containing markers tightly linked to the myotonic dystrophy locus on chromosome 19.

Myotonic dystrophy (DM) is caused by a defect in an unknown gene that maps to 19q13.3, flanked by the tightly linked markers ERCC1 on the proximal side and D19S51 on the distal side. We report the isolation and characterization of overlapping YAC and cosmid clones around D19S51 for the construction of a physical map around this locus. The resulting contig contains the markers D19S51 and D19S62 (another new marker tightly linked to the DM locus) and the distal breakpoint of a radiation hybrid cell line used in the physical mapping of the DM region. We have compared the restriction maps of the YACs and cosmids with that of the genome to investigate the fidelity of these clones.     

A 4,103 marker integrated physical and comparative map of the horse genome

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.     

High-resolution genetic map of bovine chromosome 29 through focused marker development

Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.     

Construction of bovine whole-genome radiation hybrid and linkage maps using high-throughput genotyping

High-density whole-genome maps are essential for ordering genes or markers and aid in the assembly of genome sequence. To increase the density of markers on the bovine radiation hybrid map, and hence contribute to the assembly of the bovine genome sequence, an Illumina BeadStation was used to simultaneously type large numbers of markers on the Roslin-Cambridge 3000 rad bovine-hamster whole-genome radiation hybrid panel (WGRH3000). In five multiplex reactions, 6738 sequence tagged site (STS) markers were successfully typed on the WGRH3000 panel DNA. These STSs harboured SNPs that were developed as a result of the bovine genome sequencing initiative. Typically, the most time consuming and expensive part of creating high-density radiation hybrid (RH) maps is genotyping the markers on the RH panel with conventional approaches. Using the method described in this article, we have developed a high-density whole-genome RH map with 4690 loci and a linkage map with 2701 loci, with direct comparison to the bovine whole-genome sequence assembly (Btau_2.0) in a fraction of the time it would have taken with conventional typing and genotyping methods.     

Generation and characterization of a 12,000-rad radiation hybrid panel for fine mapping in pig

We have constructed a 12,000-rad porcine whole-genome radiation hybrid panel to complement the first generation 7,000-rad panel (IMpRH) and allow higher resolution mapping studies both in specific areas of interest and on the whole genome. We analyzed 243 hybrid clones on the basis of their marker retention frequency to produce a final panel of 90 hybrid clones with an average retention frequency of 35.4%. The resolution of this 12,000-rad panel (IMNpRH2) was compared to the resolution of the 7,000-rad panel (IMpRH) by constructing framework maps in the 2.4-Mb region of porcine chromosome 15 containing the acid meat RN gene. In this region, two-point analysis was used to estimate RH distances and demonstrates their reliability with the estimation of physical distances. This study demonstrates that the 12,000-rad panel constitutes a powerful tool for constructing high-resolution maps. Indeed, the resolution of IMNpRH2 (12-14 kb/cR(12,000)) is two to three times more than that of IMpRH (35-37 kb/cR(7,000)). As expected, the increase in the radiation dose allows an increase of the mapping resolution in terms of kb/cR with the same suppleness of use for mapping experiments. In addition the RH map constructed in the region investigated proved to be more homogeneous on IMNpRH2 than on IMpRH.     

A high-resolution radiation hybrid map of the proximal region of rat Chromosome 4

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment. Received: 4 December 1998 / Accepted: 19 January 1999     

An integrated genetic linkage map of pepper (Capsicum spp.)

An integrated genetic map of pepper including 6 distinct progenies and consisting of 2262 markers covering 1832 cM was constructed using pooled data from six individual maps by the Keygene proprietary software package INTMAP. The map included: 1528 AFLP, 440 RFLP, 288 RAPD and several known gene sequences, isozymes and morphological markers. In total, 320 anchor markers (common markers in at least two individual maps) were used for map integration. Most anchor markers (265) were common to two maps, while 27, 26 and 5 markers were common to three, four and five maps, respectively. Map integration improved the average marker density in the genome to 1 marker per 0.8 cM compared to 1 marker per 2.1 cM in the most dense individual map. In addition, the number of gaps of at least 10 cM between adjacent markers was reduced in the integrated map. Although marker density and genome coverage were improved in the integrated map, several small linkage groups remained, indicating that further marker saturation will be needed in order to obtain a full coverage of the pepper genome. The integrated map can be used as a reference for future mapping studies in Capsicum and to improve the utilization of molecular markers for pepper breeding.These authors contributed equally to the work described in this paper(e-mail:     

Index, Comprehensive Microsatellite, and Unified Linkage Maps of Human Chromosome 14 with Cytogenetic Tie Points and a Telomere Microsatellite Marker

Three sets of linkage maps (index, comprehensive microsatellite, and unified) have been constructed for human chromosome 14 based on genotypes from the CEPH reference pedigrees. The index maps consist of 18 microsatellite markers, with heterozygosities of at least 68% and intermarker spacing no greater than 11 cM. The sex-average comprehensive microsatellite map is 125 cM in length and includes 115 markers with 54 loci uniquely placed with odds for marker order of at least 1000:1. The sex-average index map length is 121 cM, and the female- and male-specific maps are 143 and 101 cM, respectively. A unified map was also constructed from 147 loci (162 marker systems), which includes 32 RFLP markers in addition to the 115 microsatellites. The sex-average length of the unified map is 128 cM with 69 loci uniquely placed. Our maps are anchored by a microsatellite telomere marker sCAW1 (D14S826), developed from a telomere YAC clone TYAC196, which extends the linkage map to the physical terminus of the long arm of chromosome 14. Furthermore, we have also physically mapped seven of the loci by fluorescencein situhybridization of cosmid clones orAlu-PCR products amplified from YACs containing the marker sequences. Together with previously established cytogenetic map designations for other loci, our maps display links between genetic markers for 10 of 13 cytogenetic bands of chromosome 14 at the 550 genome band resolution.     

One hundred and one new simple sequence repeat-based markers for the canine genome

One hundred and one new dinucleotide repeat polymorphisms specific for the canine genome have been identified and characterized. Screening of both primary libraries and marker selected libraries enriched for simple sequence repeats led to the isolation of large numbers of genomic clones that contained (CA)_n repeats. Over 200 of these clones were sequenced, and PCR primers that bracket the repeat were developed for those that contained ten or more continuous (CA)_n units. This effort led to the production of 101 polymorphic markers, which were assigned to one of four categories depending on their degree of polymorphism. Fiftyfour markers were found to be highly or very highly polymorphic as they had four or more alleles when tested on a panel of unrelated dogs. This group of markers will be useful for following inheritance of traits in crosses between dogs.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers indicated in Table 1.     

A high-resolution radiation hybrid map of rhesus macaque chromosome 5 identifies rearrangements in the genome assembly

A 10,000-rad radiation hybrid (RH) cell panel of the rhesus macaque was generated to construct a comprehensive RH map of chromosome 5. The map represents 218 markers typed in 185 RH clones. The 4846-cR map has an average marker spacing of 798 kb. Alignments of the RH map to macaque and human genome sequences confirm a large inversion and reveal a previously unreported telomeric inversion. The macaque genome sequence indicates small translocations from the ancestral homolog of macaque chromosome 5 to macaque chromosomes 1 and 6. The RH map suggests that these are probably assembly artifacts. Unlike the genome sequence, the RH mapping data indicate the conservation of synteny between macaque chromosome 5 and human chromosome 4. This study shows that the 10,000-rad panel is appropriate for the generation of a high-resolution whole-genome RH map suitable for the verification of the rhesus genome assembly.     

IDENTIFICATION AND CLONING OF AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS LINKED TO THE MATING TYPE LOCUS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

Amplified fragment length polymorphism (AFLP) markers have been widely used to generate molecular maps of plant species, including crops and cereals. We report on a useful protocol to identify AFLPs from Chlamydomonas reinhardtii Dangeard with digoxigenin labeled primers. Although Chlamydomonas has a small genome with a high GC content, we could detect polymorphic bands that led to the identification of several AFLP markers linked to the mating type locus of Chlamydomonas. Three of these markers were isolated from the gel, reamplified, and cloned. The clones were sequenced, and the insertion of the correct fragment was verified in AFLP gels and in Southern blots. One marker showed sequence identity to parts of the fus1 gene, known to be unique in the plus mating type. We also converted some of the AFLP markers into sequence tagged site markers, which allows a fast and convenient screening of progeny of crosses. This procedure will be a useful and fast alternative to the conventional generation of maps for the positional cloning of genes from Chlamydomonas.     

Comparative mapping between humans and pigs: localization of 58 anchorage markers (TOASTs) by use of porcine somatic cell and radiation hybrid panels

To increase the number of Type I markers that are directly informative for comparative mapping, 58 anchorage markers, TOASTs (Traced Orthologous Amplified Sequence Tags), were mapped in pig. With specific consensus primers, 76 TOASTs were tested in pig: 50 were regionally localized in pig on a somatic cell hybrid panel (SCHP), and 51 were mapped on the whole genome, INRA/University of Minnesota porcine Radiation Hybrid panel (IMpRH). Comparison of marker positions on RH and cytogenetic maps indicated general concordance except for two chromosomal regions. For RH mapping, all markers, apart from one, were significantly linked (LOD > 4.8) to a marker of the first-generation radiation hybrid map. Localization of new markers on the initial map is necessary for drawing a framework map as shown for Chromosome Sscr 14. The addition of four TOASTs has enabled us to propose an improved map, using a threshold likelihood ratio of 1000/1. At the whole-genome level, this work significantly increased (by 50%) the number of precisely mapped genes on the porcine RH map and confirmed that the IMpRH panel is a valuable tool for high-resolution gene mapping in pig. Porcine PCR products were sequenced and compared with human sequences to verify their identity. Most of the localizations made it possible to either confirm or refine the previous comparative data between humans and pigs obtained through heterologous chromosomal painting or gene mapping. Moreover, the use of TOASTs in mapping studies appears to be a complement to other strategies using CATS, human ESTs, or heterologous FISH with BACs which had already been applied to improve the gene density of comparative genomic maps for mammals. Received: 15 March 2000 / Accepted: 27 July 2000     

Diversity arrays technology (DArT) markers in apple for genetic linkage maps

Diversity Arrays Technology (DArT) provides a high-throughput whole-genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. The work presented here details the development and performance of a DArT genotyping array for apple. This is the first paper on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high-throughput method for obtaining accurate and reproducible marker data, despite the low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. The standard complexity reduction method, based on the methylation-sensitive PstI restriction enzyme, resulted in a high frequency of markers, although there was 52-54% redundancy due to the repeated sampling of highly similar sequences. Sequencing of the marker clones showed that they are significantly enriched for low-copy, genic regions. The genome coverage using the standard method was 55-76%. For improved genome coverage, an alternative complexity reduction method was examined, which resulted in less redundancy and additional segregating markers. The DArT markers proved to be of high quality and were very suitable for genetic mapping at low cost for the apple, providing moderate genome coverage. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9579-5) contains supplementary material, which is available to authorized users.     

The construction of a tetraploid cotton genome wide comprehensive reference map

Integration of multiple genomic maps provides a higher density of markers and greater genome coverage, which not only facilitates the identification and positioning of QTLs and candidate genes, but it also provides a basic structure for the genome sequence assembly. However, the diversity in markers and populations used in individual mapping studies limits the ability to fully integrate the available data. By concentrating on marker orders rather than marker distances, published map data could be used to produce a comprehensive reference map (CRM) that includes a majority of known markers with optimally estimated order of those markers across the genome. In this study, a tetraploid cotton genome-wide CRM was constructed from 28 public cotton genetic maps. The initial CRM contained 7,424 markers and represented over 93% of the combined mapping information from the 28 individual maps. The current output is stored and displayed through CottonDB (http://www.cottondb.org), the public cotton genome database.